Difference between revisions of "Part:BBa K1189020:Experience"
 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
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how you used this part and how it worked out.
 
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===Applications of BBa_K1189020===
 
 
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<p>We evaluated the binding of our coils using other constructs that make use of the E and K coil parts submitted. In the case of the coils we were interested to see if the K-coil fused to TALE proteins (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1189029"><span class="Green"><b>BBa_K1189029</b></span></a>, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1189030"><span class="Green"><b>BBa_K1189030</b></span></a>) could bind to the E-coil found on one of our Prussian blue ferritin constructs (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1189018"><span class="Green"><b>BBa_K1189018</b></span></a>). To complete this task we placed the TALE on the membrane, washed and blocked the membrane. The ferritin protein with the complimentary coil was then added to the membrane. If this coil successfully binds to the other coil then the ferritin will not be washed off during the next wash step. We can then see if Prussian blue ferritin is bound by adding a TMB substrate solution that will cause a colour change. To this extent we saw a blue ring in this trial indicating a positive result. This suggests that our coils are actually binding in an <i>in vitro</i> system.</p>
 
  
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<p>Another interesting element of this assay is why we used two variants of the TALE K-coil negative control. A blue ring on our TALE negative control confirmed our fear that during the  second protein application and wash step that some of the ferritin with coil proteins would drift over and bind to the TALE K-coils on the nitrocellulose. This did not occur for our separate negative control (Figure 3).</p>
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<h1>Light ferritin subunit fused to an E coil</h1>
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<p>BBa_K1189020 is the light subunit of human ferritin fused to a linker E coil (<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189011">BBa_K1189011</a>), under control of a lactose inducible promoter (<a href="https://parts.igem.org/Part:BBa_R0010">BBa_R0010</a>) and a strong RBS (<a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>). Twenty-four of these light subunits will assemble to form a protein shelled, iron sequestering nanoparticle (Chasteen <i>et al.</i>, 1999) (see Figure 1). Ferritin is ubiquitous across prokaryotic and eukaryotic systems and is used to buffer intracellular iron by crystallizing it in its core using (Chasteen <i>et al.</i>, 1999).  The light ferritin purportedly contributes to nucleation to initiate iron core formation in ferritin molecules (Chasteen <i>et al.</i>, 1999).. These nanoparticles are robust, remain stable at extreme pHs, withstand temperature variations, and can be used as a protein scaffold (Kim <i>et al.</i>, 2011)</p>
  
 
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<img src="https://static.igem.org/mediawiki/2013/e/e3/UCalgary2013TRCoilbindingpreliminary.png" alt="Preliminary Coil Binding" width="757" height="751">
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<img src="https://static.igem.org/mediawiki/2013/1/18/UCalgary2013TRFerritinrender2png.png" alt="Ferritin" width="300" height="300">
 
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<p><b>Figure 3.</b> This basic qualitative assay was used to inform us whether certain elements of our system are able to bind to each other. Our TALE proteins were mounted to the membrane along with positive controls of three Prussian blue variants; two recombinant ferritins and one commercial protein. The membranes were then washed and blocked. Prussian blue ferritin with a coil was added to our TALE protein containing a coil. Prussian blue ferritin with a TALE that could bind to the DNA held by another TALE on the membrane was also added. A TMB substrate solution was added to cause a colourimetric change over 5 minutes. Positive results are indicated by dark rings of colour. Negative controls include a TALE with a coil on the same membrane and the same TALE and bovine serum albumin on separate membranes that were treated separately. Image contrast was altered to make the results more clear on a digital monitor; the same changes were applied to each element of the figure.</p>
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<p><b>Figure 1.</b> Ribbon visualization of a fully assembled ferritin protein.</p>
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<p>We also performed an immunoprecipitation assay to demonstrate the binding of the E/K coils (Figure 4).</p>
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</figure>
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<h1>Design features</h1>
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<p>BBa_K1189020 has an N-terminal fusion to an E coil connected to ferritin by a <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189026">GS linker</a> (Figure 2).</p>  
  
 
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<img src=" https://static.igem.org/mediawiki/2013/6/65/UCalgary2013TRCoilIP.png" alt="Coil Binding Immunoprecipitation Assay" width="573" height="262">
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<img src="https://static.igem.org/mediawiki/2013/thumb/4/43/BBa_K1189020_SBOL.png/800px-BBa_K1189020_SBOL.png" alt="BBa_K1189019 SBOL part figure" width="500" height="100">
 
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<p><b>Figure 4.</b> Assay showing coiled-coil interaction <i>in vitro</i>. Crude lysates from a negative control (RFP), GFP-Ecoil and His-Kcoil were combined together to investigate interaction and immunoprecipitated with GFP or an isotype control and then further probed with α-His antibody. Only in the presence of both GFP and a His tag we see a band indicating interaction.  
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<p><b>Figure 2.</b> E coil fused via a <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189026">GS linker</a> to the light ferritin subunit under control of the LacI inducible promoter and a strong RBS. This construct will for a spherical nanoparticle which can bind up to 24 proteins of interest assuming they are expressed in fusion with the complementary K coil.</p>
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</p>
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</figcaption>
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</figure>
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<p>The coil system is of utility to other iGEM teams because they can express K coils on their own proteins of interest, and bind them to the complementary E coil on ferritin. Such a coiled-coil linker system reduces potential for large protein fusions to harm ferritin formation, allowing users to build intricate nanoparticle devices with myriad proteins. See Figures 3 application examples.</p>
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<figure>
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<img src="https://static.igem.org/mediawiki/2013/0/07/UCalgary2013TRCoilflexibility.png" alt="FerriTALE Scaffold Modularity" width="800" height="219" >
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<figcaption>
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<p><b>Figure 3.</b> Using the E and K coils in combination with ferritin as a scaffold system allows the creation of brand new FerriTALEs or protein scaffolds.</a></p>
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<p>This light ferritin chain was inspired by human light ferritin (<a href="http://www.uniprot.org/uniprot/P02792">P02792 [UniParc]</a>), codon optimized for <i>E. coli K12</i>, and commercially synthesized as shown in Figure 2. The iGEM Calgary team switched this construct into pSB1C3.</p>
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<br></br>
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<h1>Results</h1>
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<p>The 2013 iGEM Calgary team did not make use of this sequence per se. Rather, they replicated this sequence using PCR, and integrated it into other constructs for their final system (<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189018">BBa_K1189018</a>, <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189021">BBa_K1189021</a>, and <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189037">BBa_K1189037</a>). Please see these respective pages for characterization data of these respective systems.</p>
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<h1>References</h1>
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<li>Chasteen, N. D., & Harrison, P. M. (1999). Mineralization in ferritin: an efficient means of iron storage. Journal of structural biology, 126(3), 182-194.</li>
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<li>Dehal, P. K., Livingston, C. F., Dunn, C. G., Buick, R., Luxton, R., & Pritchard, D. J. (2010). Magnetizable antibody-like proteins. Biotechnology journal, 5(6), 596-604.</li>
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<li>Kim, S. E., Ahn, K. Y., Park, J. S., Kim, K. R., Lee, K. E., Han, S. S., & Lee, J. (2011). Fluorescent ferritin nanoparticles and application to the aptamer sensor. Analytical chemistry, 83(15), 5834-5843.</li>
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<h1>User reviews</h1>
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===User Reviews===
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Latest revision as of 07:16, 2 November 2013

Light ferritin subunit fused to an E coil

BBa_K1189020 is the light subunit of human ferritin fused to a linker E coil (BBa_K1189011), under control of a lactose inducible promoter (BBa_R0010) and a strong RBS (BBa_B0034). Twenty-four of these light subunits will assemble to form a protein shelled, iron sequestering nanoparticle (Chasteen et al., 1999) (see Figure 1). Ferritin is ubiquitous across prokaryotic and eukaryotic systems and is used to buffer intracellular iron by crystallizing it in its core using (Chasteen et al., 1999). The light ferritin purportedly contributes to nucleation to initiate iron core formation in ferritin molecules (Chasteen et al., 1999).. These nanoparticles are robust, remain stable at extreme pHs, withstand temperature variations, and can be used as a protein scaffold (Kim et al., 2011)

Ferritin

Figure 1. Ribbon visualization of a fully assembled ferritin protein.

Design features

BBa_K1189020 has an N-terminal fusion to an E coil connected to ferritin by a GS linker (Figure 2).

BBa_K1189019 SBOL part figure

Figure 2. E coil fused via a GS linker to the light ferritin subunit under control of the LacI inducible promoter and a strong RBS. This construct will for a spherical nanoparticle which can bind up to 24 proteins of interest assuming they are expressed in fusion with the complementary K coil.

The coil system is of utility to other iGEM teams because they can express K coils on their own proteins of interest, and bind them to the complementary E coil on ferritin. Such a coiled-coil linker system reduces potential for large protein fusions to harm ferritin formation, allowing users to build intricate nanoparticle devices with myriad proteins. See Figures 3 application examples.

FerriTALE Scaffold Modularity

Figure 3. Using the E and K coils in combination with ferritin as a scaffold system allows the creation of brand new FerriTALEs or protein scaffolds.

This light ferritin chain was inspired by human light ferritin (P02792 [UniParc]), codon optimized for E. coli K12, and commercially synthesized as shown in Figure 2. The iGEM Calgary team switched this construct into pSB1C3.



Results

The 2013 iGEM Calgary team did not make use of this sequence per se. Rather, they replicated this sequence using PCR, and integrated it into other constructs for their final system (BBa_K1189018, BBa_K1189021, and BBa_K1189037). Please see these respective pages for characterization data of these respective systems.



References

  • Chasteen, N. D., & Harrison, P. M. (1999). Mineralization in ferritin: an efficient means of iron storage. Journal of structural biology, 126(3), 182-194.
  • Dehal, P. K., Livingston, C. F., Dunn, C. G., Buick, R., Luxton, R., & Pritchard, D. J. (2010). Magnetizable antibody-like proteins. Biotechnology journal, 5(6), 596-604.
  • Kim, S. E., Ahn, K. Y., Park, J. S., Kim, K. R., Lee, K. E., Han, S. S., & Lee, J. (2011). Fluorescent ferritin nanoparticles and application to the aptamer sensor. Analytical chemistry, 83(15), 5834-5843.


    • User reviews



    UNIQce5536df6305eb45-partinfo-00000001-QINU UNIQce5536df6305eb45-partinfo-00000002-QINU